The physiological importance of "oxygen-addition" reactions in various biological processes has now been widely appreciated. However, the catalytic mechanisms involved are still poorly understood. The long-term goals of our program are to further our understanding of catalytic mechanisms of microbial flavoprotein hydroxylases, a class of monooxygenase, and to elucidate the bases of regulation of hydroxylase in vitro and in vivo activities. Specifically, the present project is aimed to delineate the mechanisms, intermediates, and active sites of Beneckea harveyi luciferase and Pseudomonas cepacia salicylate hydroxylase. Both steady-state and stopped-flow kinetic techniques will be used to elucidate reaction mechanisms and intermediates of the two hydroxylases. In such studies, a mutant enzyme with altered kinetic properties, deuterated substrates, and flavin and substrate analogs will also be used as mechanistic probes. The detection, isolation, and characterization of enzyme intermediates, both those previously noted and new ones, will be pursued using cryoenxymological techniques, an approach proven fruitful in our ongoing research. The active site structures of the two hydroxylases will be elucidated by the method of photoaffinity labeling. One useful labeling reagent has already been synthesized and new ones will be developed. Studies on microbial flavo-hydroxylases have already contributed significantly to our knowledge of enzymatic activation of oxygen. The proposed investigations on salicylate hydroxylase and luciferase will advance further our understanding of the oxygen-addition reactions. Since microbial hydroxylases are important in the biodegradation of various phenolic and benzoic toxicants and pollutants, studies aimed to delineate the catalytic properties and active site structures of hydroxylases, such as those proposed in this application, will be of fundamental importance for the development of an effective regulation of hydroxylase actions for detoxification and pollutant disintegration.